Immunochromatography

ABSTRACT

An immunochromatography including steps of mixing an antigen-containable specimen and modified magnetic particles, which are magnetic particles modified with a monoclonal antibody X; collecting the magnetic particles from the mixture using magnetism; dissociating the modified magnetic particles to obtain an antigen-containing solution; neutralizing the antigen-containing solution to obtaina neutralized antigen-containing solution; spreading gold particle composite bodies on an insoluble carrier having a reaction site at which a monoclonal antibody Z has been immobilized, wherein the gold particle composite body is a composite body of the antigen and a modified gold particle which is a gold particle modified with a monoclonal antibody Y; capturing the gold particle composite bodies at the reaction site; and silver-amplifying the gold particle composite body, in which the antibody X and the antibody Y are different from each other, and the antibody X and the antibody Z are different from each other.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a Continuation of PCT International Application No.PCT/JP2020/024691 filed inn Jun. 24, 2020, which claims priority under35 U.S.C. §119 (a) to Japanese Patent Application No. 2019-178730 filedon Sep. 30, 2019. Each of the above application(s) is hereby expresslyincorporated by reference, in its entirety, into the presentapplication.

BACKGROUND OF THE INVENTION 1. Field of the invention

The present invention relates to immunochromatography.

2. Description of the Related Art

Immunochromatography is frequently used these days since the operationis easy and measurement can be carried out in a short time.

For example, in a case where an antigen such as influenza virus isdetected by immunochromatography, the following operation is carriedout.

First, a label modified with an antibody (labeled antibody) is preparedand mixed with a specimen containing an antigen. The labeled antibodybinds to an antigen, whereby composite bodies are formed. In this state,in a case where these composite bodies are spread on an insolublecarrier having a detection line (a test line) onto which an antibodythat specifically reacts with an antigen is applied, the compositebodies react with the antibody on the detection line and are captured,and detection is confirmed visually or in other manners.

Examples of such immunochromatography include the method disclosed inJP5728453B.

SUMMARY OF THE INVENTION

These days, an hnmunodiagnostic method having higher sensitivity isdesired, and regarding the immunochromatography, there is also a demandfor a method having higher sensitivity than the method in the relatedart (for example, the method disclosed in JP5728453B).

In consideration the above circumstances, an object of the presentinvention is to provide immunochromatography having high detectionsensitivity.

As a result of diligent studies on the above-described problems,inventors of the present invention have found that the above object canbe achieved by carrying out a predetermined pretreatment using magnetismand changing the kind of the monoclonal antibody that is used in eachstep,

That is, the inventors of the present invention have found that theobject can be achieved by the following configurations.

(1) Immunochromatography Comprising

a mixing step of mixing an antigen-containable specimen and modifiedmagnetic particles, which are magnetic particles modified with amonoclonal antibody X having a specific affinity to the antigen, toobtain a mixture containing magnetic particle composite bodies which arecomposite bodies of the antigen and the modified magnetic particle;

a collection step of collecting magnetic particles in the mixturecontaining the magnetic particle composite bodies using magnetism;

a dissociation step of dissociating the modified magnetic particles fromthe magnetic particle composite body to obtain an antigen-containingsolution by mixing the magnetic particles collected in the collectionstep with a dissociation solution which is an acidic or alkalinesolution;

a neutralization step of neutralizing the antigen-containing solutionusing a neutralization solution to obtain a neutralizedantigen-containing solution;

a spreading step of spreading gold particle composite bodies on aninsoluble carrier having a reaction site at which a. monoclonal antibodyZ capable of binding to the antigen has been immobilized, in a statewhere the gold particle composite body which is a composite body of theantigen in the neutralized antigen-containing solution and a modifiedgold particle which is a gold particle modified with a monoclonalantibody Y capable of binding to the antigen;

a capturing step of capturing the gold particle composite bodies at thereaction site of the insoluble carrier, and

a silver amplification step of silver-amplifying the gold particlecomposite body captured in the capturing step,

in which the monoclonal antibody X and the monoclonal antibody Y aredifferent from each other, and

the monoclonal antibody X and the monoclonal antibody Z are differentfrom each other,

(2) The immunochromatography according to (1), in which the monoclonalantibody X, the monoclonal antibody Y, and the monoclonal antibody Z areall different from each other.

(3) The immunochromatography according to (1) or (2), in which thedissociation solution is a dissociation solution which is an acidic oralkaline solution, where an amount of the dissociation solution issmaller than the antigen-containable specimen.

(4) The immunochromatography according to (3), in which a. ratio of thedissociation solution to the antigen-containable specimen is ⅕ or lessin terms of mass ratio.

(5) The immunochromatography according to any one of (1) to (4), inwhich the dissociation solution contains NaOH or HCl.

(6) The immunochromatography according to any one of (1) to (5), inwhich the neutralization solution contains HCl and at least one selectedfrom the group consisting of tricine, Tris, HEPES, acetamidoglycine,glycinamide, and vicine, or contains NaOH and at least one selected fromthe group consisting of tricine, Tris, HEPES, acetamidoglycine,glycinamide, and vicine.

(7) The immunochromatography according to any one of (1) to (6), inwhich a particle diameter of the magnetic particle before modificationis 0.05 μm to 10 μm.

As described below; according to the present invention, it is possibleto provide immunochromatography having high detection sensitivity.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic view illustrating an aspect of an insolublecarrier that is used in a method according to the embodiment of thepresent invention.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Immunochromatography according to the embodiment according to theembodiment of the present invention will be described below.

In the present specification, the numerical value range indicated byusing “to” means a range including the numerical values before and after“to” as the lower limit value and the upper limit value, respectively,

In addition, in the present specification, one kind of each componentmay be used alone, or two or more kinds thereof may be used incombination. In a case where two or more kinds of each component areused in combination, a content of the component indicates a totalcontent unless otherwise specified.

Further, in the present specification. “the detection sensitivity andthe signal/noise ratio (the S/N ratio) are further improved” is alsodescribed as “the effects and the like of the present invention are moreexcellent”.

The immunochromatography according to the embodiment of the presentinvention (hereinafter, also referred to as “the method according to theembodiment of the present invention”) is immunochromatography including;

a mixing step of mixing an antigen-containable specimen and modifiedmagnetic particles, which are magnetic particles modified with amonoclonal antibody X having a specific affinity to the antigen, toobtain a mixture containing magnetic particle composite bodies which arecomposite bodies of the antigen and the modified magnetic particle;

a collection step of collecting magnetic particles in the mixturecontaining the magnetic particle composite bodies using magnetism;

a dissociation step of dissociating the modified magnetic particles fromthe magnetic particle composite body to obtain an antigen-containingsolution by mixing the magnetic particles collected in the collectionstep with a dissociation solution which is an acidic or alkalinesolution;

a neutralization step of neutralizing the antigen-containing solutionusing a neutralization solution to obtain a neutralizedantigen-containing solution;

a spreading step of spreading gold particle composite bodies on aninsoluble carrier having a reaction site at which a monoclonal antibodyZ capable of binding to the antigen has been immobilized, in a statewhere the gold particle composite body which is a composite body of theantigen in the neutralized antigen-containing solution and a modifiedgold particle which is a gold particle modified with a monoclonalantibody Y capable of binding to the antigen;

a capturing step of capturing the gold particle composite bodies at thereaction site of the insoluble carrier, and

a silver amplification step of silver-amplifying the gold particlecomposite body captured in the capturing step,

in which the monoclonal antibody X and the monoclonal antibody Y aredifferent from each other, and.

the monoclonal antibody X and the monoclonal antibody Z are differentfrom each other.

As described above, in the method according to the embodiment of thepresent invention, the predetermined pretreatment (the mixing step, thecollection step, the dissociation step, and the neutralization step)using magnetism is carried out, and the kind of the monoclonal antibodythat is used in a specific step is changed to a different kind.

It is presumed that since the method according to the embodiment of thepresent invention has such a configuration, the above effects can beobtained. In particular, it is conceived that the method of the presentinvention is characterized in that the monoclonal antibody X that isused in the above pretreatment is different from the monoclonal antibodyY and/or monoclonal antibody Z that is used in the spreading step.

From the studies by inventors of the present invention, it has beenknown that in a case where an antigen is reacted with magnetic particles(modified magnetic particles) modified with a monoclonal antibody, andafter an operation such as concentration, the modified magneticparticles are dissociated. to obtain an antigen-containing solution(particularly, an antigen-concentrated solution), there is a case wherethe dissociated modified magnetic particles remain in the solution andinhibit the antigen-antibody reaction in the immunochromatography.

The method according to the embodiment of the present invention is basedon the above findings. That is, in the method according to theembodiment of the present invention, the monoclonal antibody that isused in the above pretreatment and the monoclonal antibody that is usedin the immunochromatography (the spreading step) are different from eachother. As a result, it is presumed that even in a case where thedissociated modified magnetic particles (the magnetic particles modifiedwith a monoclonal antibody) react with the antigen again, a highdetection sensitivity can be obtained without inhibiting theantigen-antibody reaction in the immunochromatography.

Hereinafter, each of the steps included in the method according to theembodiment of the present invention will be described. It is noted thatthe steps from the mixing step to the neutralization step are alsocollectively referred to as a “magnetic particle process”.

Mixing Step

The mixing step is a step of mixing an antigen-containable specimen andmodified magnetic particles, which are magnetic particles modified witha monoclonal antibody X having a specific affinity to the antigen, toobtain a mixture containing magnetic particle composite bodies which arecomposite bodies of the antigen and the modified magnetic particle.

Specimen

The specimen that is used in the mixing step is not particularly limitedas long as it is an antigen-containable specimen. Examples of such aspecimen include a biological specimen, particularly a biologicalspecimen of animal origin (particularly, of human origin) such as a bodyfluid (for example, blood, serum, plasma, spinal fluid, tear fluid,sweat, urine, pus, runny nose, or sputum) or excrement (for example,feces), an organ, a tissue, a mucous membrane or skin, a scraped testsample (a swab) that is conceived to contain these substances, amouthwash, and an animal and a plant themselves or a dried substancethereof.

Antigen

Examples of the antigen include a fungus, a bacterium (for example,tubercle bacillus or lipoarabinomannan (LAM) included in the tuberclebacillus), a virus (for example, an influenza virus), and a nuclearprotein thereof LAM is a major antigen in tuberculosis and a glycolipidwhich is a major constitutional component of the cell membrane and thecell wall.

The antigen is preferably a virus or LAM, more preferably a virus, andstill more preferably an influenza virus, due to the reason that theeffects and the like of the present invention are more excellent.

Pretreatment of Specimen

Regarding the above specimen, it is possible to use the specimen as itis or in a form of an extraction solution obtained by extracting theantigen using an appropriate solvent for extraction, in a form of adiluent solution obtained by diluting an extraction solution with anappropriate diluent, or in a fomi in which an extraction solution hasbeen concentrated by an appropriate method.

As the solvent for extraction, it is possible to use a solvent (forexample, water, physiological saline, and a buffer solution) that isused in a general immunological analysis method, or a water-miscibleorganic solvent with which a direct antigen-antibody reaction can becarried out by being diluted with such a solvent.

Modified Magnetic Particle

The modified magnetic particle is a magnetic particle modified with themonoclonal antibody X having a specific affinity to the antigen.

Magnetic Particle

A material of the magnetic particles is not particularly limited as longas it is a material having magnetic properties, and specific examplesthereof include iron, cobalt, nickel, oxides thereof, ferrite, alloysthereof. Among them, iron oxide is preferable due to the reason that theeffects and the like of the present invention are more excellent.

The magnetic particle may be a particle obtained by molding only amaterial having magnetic properties into a particle shape.Alternatively, the magnetic particle may be a particle of which thesurface has been coated with a polymer (such as polystyrene or silicagel) or the like and which has a material having magnetic properties asa core, or may be a particle of which the surface has been coated usinga material having magnetic properties and which has a polymer or thelike as a core.

Particle Diameter

The particle diameter of the magnetic particle is not particularlylimited; however, it is preferably 0.05 μm to 10 μm. and more preferablyto 0.1 μm to 5 μm due to the reason that the effects and the like of thepresent invention are more excellent.

The particle diameter can be measured with a commercially availableparticle diameter distribution meter or the like. As a method ofmeasuring the particle size distribution, optical microscopy, confocallaser microscopy, electron microscopy, atomic force microscopy, staticlight scattering method, laser diffraction method, dynamic lightscattering method, centrifugal sedimentation method, electric pulsemeasurement method, chromatography method, ultrasonic attenuationmethod, and the like are known, and apparatuses corresponding to therespective principles are commercially available. As the method ofmeasuring a particle diameter, a dynamic light scattering method can bepreferably used due to the particle diameter range and the ease ofmeasurement. Examples of the commercially available measuring deviceusing dynamic light scattering include NANOTRAC UPA (Ni.kkiso Co.,Ltd.), a dynamic light scattering type particle size distributionmeasuring device LB-550 (HORIBA, Ltd.), and a Fiber-Optics ParticleAnalyzer FPAR-I000 (Otsuka Electronics Co., Ltd.). In the presentinvention, the average particle size is obtained as a value of a mediandiameter (d=50) measured at a measurement temperature of 25° C.

Monoclonal Antibody X:

The monoclonal antibody X is a monoclonal antibody having a specificaffinity to the above antigen and is a monoclonal antibody differentfrom the monoclonal antibody Y and the monoclonal antibody Z describedlater.

As the monoclonal antibody, it is possible to use, for example, amonoclonal antibody obtained by cell fusion using spleen cells of ananimal immunized with an antigen, or a fragment thereof [for example,F(ab′)₂, Fab, Fab′, or Fv]. The preparation of these antibodies can becarried out by a conventional method.

Method of Manufacturing Modified Magnetic Particle

The method of manufacturing the modified magnetic particle is notparticularly limited, and a known method can be used. Examples thereofinclude a method of activating magnetic particles with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) to carry themonoclonal antibody X on the magnetic particle.

Mixing

In the mixing step, the specimen and the modified magnetic particles aremixed.

As a result, in a case where the specimen contains an antigen, theantigen in the specimen reacts with the monoclonal antibody X having aspecific affinity to the antigen of the modified magnetic particle, andthereby a composite body of the antigen and the modified magneticparticle is formed in the specimen. On the other hand, in a case wherethe specimen does not contain an antigen, the composite body is notformed.

Collection Step

The collection step is a step of collecting magnetic particles in themixture containing the magnetic particle composite body after theahove-described mixing step by using magnetism.

Here, “collecting magnetic particles in the mixture” means “collectingthe magnetic particle composite bodies in the mixture as well as themagnetic particles (unmodified magnetic particles) and the modifiedmagnetic particles remaining in the mixture”.

The method of collecting magnetic particles in the mixture after themixing step by using magnetism is not particularly limited. Examplesthereof include a method of placing the mixture after the mixing step ina conical tube installed on a magnetic stand, collecting the magneticparticle composite bodies with magnetism, and then removing the excessspecimen.

Dissociation Step

The dissociation step is a step of dissociating the modified magneticparticles from the magnetic particle composite body to obtain anantigen-containing solution by mixing the magnetic particles collectedin the collection step described above with a dissociation solutionwhich is an alkaline or acidic solution.

In the dissociation step, the modified magnetic particles aredissociated from the magnetic particle composite bodies (the compositebodies of the antigen and the modified magnetic particles) by thedissociation solution, and they are separated into the antigen and themodified magnetic particles. As a result, an antigen-containing solutionthat is a dissociation solution containing the antigen is obtained.

Dissociation Solution

The dissociation solution is not particularly limited as long as it isan alkaline or acidic solution.

The alkaline solution is not particularly limited; however, specificexamples thereof include an aqueous NaOH solution and an aqueous KOHsolution. Among them, an aqueous NaOH solution is preferable due to thereason that the effects and the like of the present invention are moreexcellent.

The acidic solution is not particularly limited; however, specificexamples thereof include an aqueous HCl solution, an aqueous H₂SO₄solution, and an aqueous HNO₃ solution, Among them, an aqueous HClsolution is preferable due to the reason that the effects and the likeof the present invention are more excellent.

The dissociation solution is preferably an alkaline solution due to thereason that the effects and the like of the present invention are moreexcellent.

The dissociation solution preferably contains NaOH or HCl_(—) morepreferably contains NaOH, and still more preferably an aqueous NaOHsolution, due to the reason that the effects and the like of the presentinvention are more excellent.

Amount

The amount of the dissociation solution is not particularly limited;however, it is preferably an amount of the dissociation solution, whichsmaller than the amount of the “antigen-containable specimen” that isused in the above-described mixing step due to the reason that theeffects and the like of the present invention are more excellent. In acase where the amount of the dissociation solution is smaller than theamount of the “antigen-containable specimen” that is used in theabove-described mixing step, the concentration of the antigen in thedissociation solution is higher than the concentration of the antigen inthe “antigen-containable specimen” that is used in the above-describedmixing step. That is, the antigen-containing solution obtained by thedissociation step is a solution (an antigen-concentrated solution) inwhich the concentration of the antigen is concentrated.

The ratio of the dissociation solution to the “antigen-containablespecimen” that is used in the above-described mixing step is preferably⅕ or less, more preferably 1/10 or less, and still more preferably 1/20or less in terms of mass ratio due to the reason that the effects andthe like of the present invention are more excellent.

Neutralization Step

The neutralization step is a step of neutralizing the antigen-containingsolution obtained in the above-described dissociation step by using aneutralization solution to obtain a neutralized antigen-containingsolution.

The antigen-containing solution obtained in the dissociation step isusually alkaline or acidic since a dissociation solution which is analkaline or acidic solution is used for the dissociation in theabove-described dissociation step. On the other hand, in a case where analkaline or acidic solution is used in the spreading step describedlater, the monoclonal antibody Y or monoclonal antibody Z describedlater may be modified, which leads to a decrease in detectionsensitivity. For this reason, in the neutralization step, theneutralization solution is used to neutralize the antigen-containingsolution.

Neutralization Solution

The neutralization solution is not particularly limited; however, it ispossible to use, for example, a known buffer solution.

Due to the reason that the effects and the like of the present inventionare more excellent, in a case where the dissociation solution that isused in the above-described dissociation step is an alkaline solution,the neutralization solution is preferable to contain HCl (particularly,1M HCl) and at least one selected from the group consisting of tricine,Tris. 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES),acetamidoglycine, glycinamide, and vicine, and in a case where thedissociation solution used in the dissociation step described above isan acidic solution, the neutralization solution is preferable to containNaOH (particularly, 1M NaOH) and at least one selected from the groupconsisting of trieine, Tris, HEPES, acetamidoglycine, glycinamide, andvicine,

Spreading Step

The spreading step is a step of spreading gold particle composite bodieson an insoluble carrier having a reaction site at which a monoclonalantibody Z capable of binding to an antigen in the neutralizedantigen-containing solution obtained in the neutralization stepdescribed above has been immobilized, in a state where the gold particlecomposite bodies which are composite bodies of the antigen and modifiedgold particles which are gold particles modified with a monoclonalantibody Y capable of binding to the antigen are formed.

Gold Particle Composite Body

As described above, in the spreading step, first, the gold particlecomposite body which is a composite body of the antigen in theneutralized antigen-containing solution obtained in the above-describedneutralization step and a modified gold particle which is a goldparticle modified with the monoclonal antibody Y capable of binding tothe antigen is formed.

Modified Gold Particle

The modified gold particle is a gold particle modified with themonoclonal antibody Y capable of binding to an antigen.

Gold Particle

Gold particle is not particularly limited.

The gold particle acts as a catalyst that reduces silver ions in thesilver amplification step described later.

The particle diameter of the gold particles is preferably 100 am orless, more preferably 50 nm or less, still more preferably 30 nm orless, and particularly preferably 15 nm or less, due to the reason thatthe effects and the like of the present invention are more excellent.

The lower limit of the particle diameter of the gold particles is notparticularly limited; however, it is preferably 1 am or more, morepreferably 2 nm or more, and still more preferably 5 nm or more, due tothe reason that the effects and the like of the present invention aremore excellent.

The particle diameter of the gold particle can be determined by the samemethod as the method for the magnetic particles described above.

Monoclonal Antibody Y

The monoclonal antibody Y is a monoclonal antibody capable of binding tothe above antigen and is not particularly limited as long as it is amonoclonal antibody different from the above monoclonal antibody X.

Specific examples and the preferred aspect of the monoclonal antibody Yare the same as those of the monoclonal antibody X described above.

Method of Manufacturing Modified Gold Particle

The method of manufacturing the modified gold particle is notparticularly limited, and a known method can be used. Examples thereofinclude a chemical bonding method such as a method in which an SH groupis introduced into the monoclonal antibody Y, amd the fact that gold andan SH group are chemically bonded is utilized so that the SH bond of theantibody is cleaved to generate an Au—S bond on the Au surface when theantibody approaches gold particles, whereby the antibody is immobilized.

Insoluble Carrier

The above-described insoluble carrier is an insoluble carrier having areaction site (a test line) at which the monoclonal antibody Z capableof binding to the antigen is immobilized. The insoluble carrier may havea plurality of test lines depending on the kinds of antigens (forexample, a test line for influenza A type virus and a test line forinfluenza B type virus). In addition, the insoluble carrier may have acontrol line on the downstream side of the test line in order to checkthe spreading of the gold particle composite bodies. Further in a casewhere a reducing agent solution is used in the silver amplification stepdescribed later, a coloring reagent immobilization line may be provideddownstream of the test line in order to detect the reducing agentsolution.

Examples of the specific aspect of the insoluble carrier include anitrocellulose membrane 100 as illustrated in FIG. 1, which has from theupstream side; a gold colloid holding pad 10, a test line 20, a controlline 30, and a coloring reagent immobilization line 40, Here, the goldcolloid holding pad 10 is a pad that holds gold particles (modified goldparticles) modified with the monoclonal antibody Y, the test line 20 isa line on which the monoclonal antibody Z is immobilized, the controlline 30 is a line for checking the spreading, and the coloring reagentimmobilization line 40 is a line for detecting the reducing agentsolution described later. Here, the upstream side and the downstreamside mean descriptions intended to indicate the spreading from theupstream side to the downstream side at the time when gold particlecomposite bodies are spread.

The specific aspect of the insoluble carrier (or animmunochromatographic kit having the insoluble carrier) includes theinsoluble carrier or the immunochromatographic kit disclosed inJP5728453B. In addition, the entire content of JP5728453B relating tothe insoluble carrier and the immunochromatographic kit is incorporatedin the present specification as a part of the disclosure of the presentspecification.

Insoluble Carrier

The insoluble carrier is preferably a porous carrier. In particular, dueto the reason that the effects and the like of the present invention aremore excellent, it is preferably a nitrocellulose film (a nitrocellulosemembrane), a cellulose membrane, an acetyl cellulose membrane, apolysulfone membrane, a polyether sulfone membrane, a nylon membrane, aglass fiber, a non-woven fabric, a cloth, a thread, or the like ispreferable, and a nitrocellulose film is more preferable.

Monoclonal Antibody Z

The monoclonal antibody Z is a monoclonal antibody capable of binding tothe above antigen and is not particularly limited as long as it is amonoclonal antibody different from the above monoclonal antibody X.

Specific examples and the preferred aspect of the monoclonal antibody Zare the same as those of the monoclonal antibody X described above.

Spreading

The method of spreading gold particle composite bodies on an insolublecarrier having a test line in a state where the gold particle compositebodies are formed is not particularly limited; however, examples thereofinclude a method in which the above nitrocellulose membrane 100 (or animmunochromatographic kit having the nitrocellulose membrane 100) asillustrated in FIG. 1 is prepared, and the neutralizedantigen-containing solution obtained in the above-describedneutralization step is dropwise added onto a gold colloid holding padand moved from the upstream side to the downstream side by using thecapillary phenomenon as illustrated in FIG. 1.

Capturing Step

The capturing step is a step of capturing the gold particle compositebodies at the reaction site of the insoluble carrier.

As described above, since the monoclonal antibody Z capable of bindingto an antigen is immobilized at the reaction site of the insolublecarder, the gold particle composite bodies (the composite bodies of anantigen and modified gold particles) spread on the insoluble carrier inthe spreading step is captured at the reaction site (the test line) ofthe insoluble carrier.

In a case where a specimen does not contain an antigen, the goldparticle composite body is not formed, and thus the composite body isnot captured at the reaction site of the insoluble carrier.

Silver Amplification Step

The silver amplification step is a step of silver-amplifying the goldparticle composite body captured in the capturing step.

The silver amplification step is a step of forming large silverparticles in the gold particle composite body captured at the reactionsite of the insoluble carrier by providing silver ions to the insolublecarrier after the capturing step. More specifically, it is a step inwhich silver ions are reduced using gold particles of the gold particlecomposite body as a catalyst to form silver particles (for example, adiameter of 10 μm or more).

This significantly improves the detection sensitivity of the capturedgold particle composite body.

Suitable Aspect

The method of providing silver ions to the insoluble carrier after thecapturing step is not particularly limited; however, it is preferably amethod in which the following reducing agent solution and the followingsilver amplification solution are used, due to the reason that theeffects and the like of the present invention are more excellent.

Further, in addition to the reducing agent solution and the silveramplification solution, a washing solution may be used to wash thecomposite body remaining on the insoluble carrier except for thespecific binding reaction. The reducing agent solution may also serve asa washing solution.

Reducing Agent Solution

The reducing agent solution contains a reducing agent capable ofreducing silver ions, As the reducing agent capable of reducing silverions, any inorganic or organic material or a mixture thereof can be usedas long as it can reduce silver ions to silver. Preferred examples ofthe inorganic reducing agent include a reducing metal salt and areducing metal complex salt, of which the atomic valence is capable ofbeing changed with a metal ion such as Fe²⁺, V²⁺, or Ti³⁺. In a casewhere an inorganic reducing agent is used, it is necessary to remove ordetoxify oxidized ions by complexing or reducing the oxidized ions. Forexample, in a system in which Fe²⁺is used as the reducing agent, acomplex of Fe³⁺, which is an oxide, is formed using citric acid orethylenediaminetetraacetic acid (EDTA), and therefore detoxification ispossible. In the present invention, it is preferable to use such aninorganic reducing agent, and as a more preferable aspect of the presentinvention, it is preferable to use a metal salt of Fe²⁺ as the reducingagent.

It is also possible to use, as the reducing agent, a main developingagent (for example, methyl gallate, hydroquinone, substitutedhydroquinone, 3-pyrazolidones, p-aminophenols, p-phenylenediamines,hindered phenols, amidoximes, azines, catechols, pyrogallols, ascorbicacid (or derivatives thereof), or leuco dyes) that is used in a wet-typelight-sensitive silver halide photographic material, and other materialsobvious to those who are skilled in the technology in the present field,such as a material disclosed in U.S. Pat. No. 6,020,117A.

As the reducing agent, an ascorbic acid reducing agent is alsopreferable. The useful ascorbic acid reducing agent includes ascorbicacid, an analog thereof, an isomer thereof, and a derivative thereof.Preferred examples thereof include D- or L-ascorbic acid and a sugarderivative thereof (for example, γ-lactoascorbic acid, glucoascorbicacid, fucoascorbic acid, glucoheptoascorbic acid, or maltoascorbicacid), a sodium salt of ascorbic acid, a potassium salt of ascorbicacid, isoascorbic acid (or L-erythroascorbic acid), a salt thereof (forexample, an alkali metal salt, an ammonium salt, or a salt known in therelated technical field), ascorbic acid of the enediol type, ascorbicacid of the enaminol type, ascorbic acid of the thioenol type.Particularly preferred examples thereof include D-, L-, or D,L-ascorbicacid (and an alkali metal salt thereof) or isoascorhic acid (or analkali metal salt thereof), and a sodium salt is a preferred salt. Amixture of these reducing agents can be used as necessary.

Due to the reason that the effects and the like of the present inventionare more excellent, the reducing agent solution is preferably allowed toflow so that the angle between the spreading direction in the spreadingstep and the spreading direction of the reducing agent solution is 0degrees to 150 degrees, and more preferably allowed to flow so that theangle between the spreading direction in the spreading step and thespreading direction of the reducing agent solution is 0 degrees to 135degrees.

Examples of the method of regulating the angle between the spreadingdirection in the spreading step and the spreading direction of thereducing agent solution include the method described in Examples ofJP2009-150869A.

Silver Amplification Solution

The silver amplification solution is a solution containing a compoundcontaining silver ions. As the compound containing silver ions, it ispossible to use, for example, organic silver salts, inorganic silversalts, or silver complexes. Preferred examples thereof include silverion-containing compounds having a high solubility in a solvent such aswater, such as silver nitrate, silver acetate, silver lactate, silverbutyrate, and silver thiosulfate. Silver nitrate is particularlypreferable. The silver complex is preferably a silver complex in whichsilver is coordinated with ligands having a water-soluble group such asa hydroxyl group or a sulfone group, and examples thereof include silverhydroxythioether.

As the silver, the organic silver salt, the inorganic silver salt, orthe silver complex is preferably contained in the silver amplificationsolution at a concentration of 0.001 mol/L to 5 mol/L, preferably 0.005mol/L to 3 mol/L, and more preferably 0.01 mol/L to 1 mol/L.

Examples of the auxiliary agent of the silver amplification solutioninclude a buffer, a preservative such as an antioxidant or an organicstabilizer, and a rate regulating agent. As the buffer, it is possibleto use, for example, a buffer formed of acetic acid, citric acid, sodiumhydroxide, or one of salts of these compounds, or formed oftris(hydroxymethyl)aminomethane, or other buffers that are used ingeneral chemical experiments. These buffers are appropriately used toadjust the pH of the amplification solution to an optimum pH thereof. Inaddition, as the antifogging agent, an alkyl amine can be used as anauxiliary agent, and dodecyl amine is particularly preferable. Inaddition, a surfactant can be used for the intended purpose of improvingthe solubility of this auxiliary agent, and C₉H₁₉—C₆H₄—O—(CH₂CH₂O)₅₀H isparticularly preferable.

Due to the reason that the effects and the like of the present inventionare more excellent, the silver amplification solution is preferablyallowed to flow from the direction opposite to the spreading directionin the spreading step described above and more preferably allowed toflow so that the angle between the spreading direction in the spreadingstep and the spreading direction of the silver amplification solution is45 degrees to 180 degrees.

Examples of the method of regulating the angle between the spreadingdirection in the spreading step and the spreading direction of thesilver amplification solution include the method described in Examplesof JP2009-150869A.

Monoclonal Antibody

As described above, the monoclonal antibody X and the monoclonalantibody Y are different from each other; and the monoclonal antibody Xand the monoclonal antibody Z are different from each other.

The monoclonal antibody Y and the monoclonal antibody Z may be the sameor different from each other; however, they are preferably differentfrom each other due to the reason that the effects and the like of thepresent invention are more excellent. In this case, the monoclonalantibody X, the monoclonal antibody Y, and the monoclonal antibody Z areall different monoclonal antibodies.

It is preferable that the epitope of the monoclonal antibody X and theepitope of the monoclonal antibody Y are different from each other dueto the reason that the effects and the like of the present invention aremore excellent.

It is preferable that the epitope of the monoclonal antibody X and theepitope of the monoclonal antibody Z are different from each other dueto the reason that the effects and the like of the present invention aremore excellent.

The epitope of the monoclonal antibody Y and the epitope of themonoclonal antibody Z may be the same or different from each other;however, they are preferably different from each other due to the reasonthat the effects and the like of the present invention are moreexcellent. In this case, the epitope of the monoclonal antibody X, theepitope of the monoclonal antibody Y, and the epitope of the monoclonalantibody 7 are all different.

The difference in epitope between monoclonal antibodies can be confirmedby, for example, an enzyme-linked immuno-sorbent assay (ELISA).

EXAMPLES

Hereinafter, the present invention will be described in more detail withreference to Examples; however, the present invention is not limitedthereto.

Preparation of Sample Solution

A sample solution (an antigen-containable specimen) was prepared using aQuick A-B “SEIKEN” negative/positive control solution (product number:322968, manufactured by DENKA SEIKEN Co., Ltd.).

Specifically, the above positive control solution was subjected toserial 10-time dilutions with a phosphate buffered salts (PBS) buffercontaining 1% by mass bovine serum albumin (BSA), and sample solutions(antigen-containable specimens) with the respective dilution rates wereprepared.

The detection limit (the minimum detection sensitivity) obtained by thecommercially available immunochromatographic detection kit “Capilia FluA+B” (manufactured by Alfresa Pharma Corporation) was 1/40 for both Atype and B type.

Example 1

Immunochromatography of Example 1 was carried out as follows.

Mixing Step

Magnetic particles (Dynabeads MyOne-COOH, particle diameter: 1 μm,manufactured by Thermo Fisher Scientific, Inc.) manufactured by ThermoFisher Scientific, Inc. were activated with1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC), and ananti-influenza type A monoclonal antibody (Funakoshi Co., Ltd.,#GTX629544) was carried on the magnetic particles to obtain modifiedmagnetic particles which are magnetic particles modified with theanti-influenza. A type monoclonal antibody (the monoclonal antibody X).

The above sample solution (6 mL) was placed in a 15 mL conical tube, Img of the above modified magnetic particles were placed therein, and thereaction was carried out with stirring for 40 minutes. in this manner, amixture containing magnetic particle composite bodies, which arecomposite bodies of the influenza type A virus and the modified magneticparticles, was obtained.

Collection Step

After the reaction, the conical tube was installed on a magnet stand,and the magnetic particles (unmodified magnetic particles, modifiedmagnetic particles, and magnetic particle composite bodies) weremagnetically collected for 10 minutes. The excess sample solution wasremoved from the conical tube with a pipette (manufactured by Eppendorf)to collect the above magnetic particles (unmodified magnetic particles,modified magnetic particles, and magnetic particle composite bodies).

Dissociation Step

Immediately, 200 μL of an aqueous 50 mM NaOH solution (an alkalinesolution) was added. After the addition, sonication treatment wascarried out for 5 minutes followed by being allowed to stand for 60minutes. In this manner, the modified magnetic particles weredissociated from the magnetic particle composite bodies. After beingallowed to stand, the conical tube was installed on a magnet standagain, and the dissociated modified magnetic particles were magneticallycollected for 10 minutes, and the supernatant solution (the influenza Atype virus-containing solution) (the antigen-containing solution) wasrecovered.

Neutralization Step

The influenza A type virus-containing solution was neutralized using 10μL of 10 mM Tris HCl as the neutralization solution to obtain aneutralized influenza A type virus-containing solution (a neutralizedantigen-containing solution).

Spreading Step

The nitrocellulose membrane 100 as illustrated in FIG. 1 was prepared,which has from the upstream side; the gold colloid holding pad 10, thetest line 20, the control line 30, and the coloring reagentimmobilization line 40. The gold colloid holding pad 10 is a pad thatholds gold colloids (modified gold particles) modified with ananti-influenza A type monoclonal antibody (Anti-Influenza A SPTN-5 7307,Medix Biochemica) (the monoclonal antibody Y), the test line 20 is aline on which the anti-influenza A type monoclonal antibody(Anti-Influenza A SPTN-5 7307, Mcdix Biochemical) (the monoclonalantibody Z) is immobilized, the control line 30 is a line for checkingthe spreading, and the coloring reagent immobilization line 40 is a linefor detecting the reducing agent solution described later.

The above-described neutralized influenza A type virus-containingsolution was dropwise added onto the gold colloid holding pad. As aresult, gold particle composite bodies, which are composite bodies ofthe influenza A type virus in the solution and the gold colloidparticles (modified gold particles) modified with the anti-influenza Atype monoclonal antibody (the monoclonal antibody Y) in the gold colloidholding pad, were formed. In this state, the gold particle compositebodies were spread toward the downstream side of the nitrocellulosemembrane.

Capturing Step

The gold particle composite.bodies that are spread in the spreading stepis captured on the test line.

Silver Amplification Step

The silver amplification step was carried out as follows.

Preparation of Reducing Agent Solution

23.6 mL of an aqueous solution of 1 mol/L iron nitrate, which wasproduced by dissolving iron (III) nitrate nonahydrate (manufactured byFUJIFILM Wako Pure Chemical Corporation) in water, and 13.1 g of citricacid (manufactured by FUJIFILM Wako Pure Chemical Corporation) weredissolved in 290 g of water. After all of the substances were dissolved,36 mL of nitric acid (10% by mass) was added thereto while stirring witha stirrer, 60.8 g of ammonium iron (II) sulfate hexahydrate(manufactured by FUJIFILM Wako Pure Chemical Corporation) was addedthereto, and the resultant solution was used as the reducing agentsolution.

Preparation of Silver Amplification Solution

8 mL of a silver nitrate solution (including 10 g of silver nitrate) and24 mL of an aqueous solution of 1 MOH, iron nitrate were added to 66 gof water. Further, this solution was mixed with a solution obtained bydissolving 5.9 mL of nitric acid (10% by mass), 0.1 g of dodecyl amine(manufactured by FUJIFILM Wako Pure Chemical Corporation), and 0.1 of asurfactant C₁₂H₂₅-C₆H₄—O—(CH₂CH₂O)₅₀H in 47.6 g of water in advance, andthe resultant solution was used as the silver amplification solution.

Spreading of Reducing Agent Solution

In the nitrocellulose membrane, the reducing agent solution prepared asdescribed above was allowed to flow from the same direction as that ofthe spreading step described above (from the upstream side).

Spreading of Silver Amplification Solution

After the coloring reagent immobilization line was discolored, thesilver amplification solution prepared as described above was allowed toflow from the direction opposite to the spreading direction (from thedownstream side) in the spreading step. In this manner, the goldparticle composite body captured on the test line was silver amplified.

Evaluation

The coloration of the test line was visually checked, and the lowestdilution rate (the minimum detection sensitivity) at which colorationwas confirmed was investigated. The results are shown in Table 1. Itmeans that as the minimum detection sensitivity is lower, an antigen canbe detected even in a specimen having a low antigen concentration, whichmeans the detection sensitivity is high.

Comparative Example

Immunochromatography was carried out and evaluated according to the sameprocedure as in Example 1 except that without carrying out the stepsfrom the mixing step to the neutralization step, the above-describedsample solution itself was used instead of the neutralized influenza Atype virus-containing solution in the spreading step. The results areshown in Table 1.

Comparative Example 2

Immunochromatography was carried out and evaluated according to the sameprocedure as in Example 1 except that as the monoclonal antibody X, ananti-influenza A type monoclonal antibody (Anti-Influenza A SPTN-5 7307,Mcdix Biochemica) was used instead of the anti-influenza A typemonoclonal antibody (Funakoshi Co., Ltd, #GTX629544 ). The results areshown in Table 1. In Comparative Example 2, the monoclonal antibody Xand the monoclonal antibody Y are the same, and the monoclonal antibodyX and the monoclonal antibody Z are the same.

TABLE 1 Comparative Comparative Example 1 Example 2 Example 1 Minimum1/20 1/1600 1/4800 detection sensitivity

As can be seen from Table 1, as compared with Comparative Example 1 inwhich the magnetic particle process was not carried out and ComparativeExample 2 in which the magnetic particle process was carried out and themonoclonal antibody X and the monoclonal antibody Y were the same andthe monoclonal antibody X and the monoclonal antibody Z were the same,Example 1 in which the magnetic particle process was carried out but themonoclonal antibody X and the monoclonal antibody Y were different fromeach other and the monoclonal antibody X and the monoclonal antibody Zwere different from each other exhibited high detection sensitivity.

In addition, when immunochromatography was carried out in the samemanner as in Example 1 and Comparative Examples 1 and 2 described aboveexcept that as the monoclonal antibody (the monoclonal antibody X, themonoclonal antibody 1, or the monoclonal antibody Z), an anti-influenzaB type monoclonal antibody was used instead of the anti-influenza A typemonoclonal antibody, the same tendency as in Table I was observed.

EXPLANATION OF REFERENCES

10: gold colloid holding pad

20: test line

30: control line

40 coloring reagent immobilization line

100: nitrocellulose membrane

What is claimed is:
 1. Immunochromatography comprising: mixing anantigen-containable specimen and modified magnetic particles, which aremagnetic particles modified with a monoclonal antibody X having aspecific affinity to the antigen, to obtain a mixture containingmagnetic particle composite bodies which are composite bodies of theantigen and the modified magnetic particle; collecting magneticparticles in the mixture containing the magnetic particle compositebodies using magnetism; dissociating the modified magnetic particlesfrom the magnetic particle composite body to obtain anantigen-containing solution by mixing the collected magnetic particleswith a dissociation solution which is an acidic or alkaline solution;neutralizing the antigen-containing solution using a neutralizationsolution to obtain a neutralized antigen-containing solution; spreadinggold particle composite bodies on an insoluble carrier having a reactionsite at which a monoclonal antibody Z capable of binding to an antigenin the neutralized antigen-containing solution has been immobilized, ina state where the gold particle composite bodies which are compositebodies of the antigen and modified gold particles which are goldparticles modified with a monoclonal antibody Y capable of binding tothe antigen are formed; capturing the gold particle composite bodies atthe reaction site of the insoluble carrier; and silver-amplifying thecaptured gold particle composite body, wherein the monoclonal antibody Xand the monoclonal antibody Y are different from each other, and themonoclonal antibody X and the monoclonal antibody Z are different fromeach other.
 2. The immunochromatography according to claim 1, whereinthe monoclonal antibody X, the monoclonal antibody Y and the monoclonalantibody Z are all different from each other.
 3. Theimmunochromatography according to claim 1, wherein the dissociationsolution is a dissociation solution which an acidic or alkalinesolution, where an amount of the dissociation solution is smaller thanthe antigen-containable specimen.
 4. The immunochromatography accordingto claim
 2. wherein the dissociation solution is a dissociation solutionwhich is an acidic or alkaline solution, where an amount of thedissociation solution is smaller than the antigen-containable specimen.5. The immunochromatography according to claim 3, wherein a ratio of thedissociation solution to the antigen-containable specimen is ⅕ or lessin terms of mass ratio.
 6. The immunochromatography according to claim4, wherein a ratio of the dissociation solution to theantigen-containable specimen is ⅕ or less in terms of mass ratio.
 7. Theimmunochromatography according to claim 1, wherein the dissociationsolution contains NaOH or HCl.
 8. The immunochromatography according toclaim 2, wherein the dissociation solution contains NaOH or HCl.
 9. Theimmunochromatography according to claim 3, wherein the dissociationsolution contains NaOH or HO,
 10. The immunochromatography according toclaim 4, wherein the dissociation solution contains NaOH or HCl.
 11. Theimmunochromatography according to claim 1, wherein the neutralizationsolution contains HCl and at least one selected from the groupconsisting of tricine, Tris. HEPES, acetamidoglycine, glycinami de, andvicine, or contains NaOH and at least one selected from the groupconsisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide, andvicine.
 12. The immunochromatography according to claim 2, wherein theneutralization solution contains HCl and at least one selected from thegroup consisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide,and vicine, or contains NaOH and at least one selected from the groupconsisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide, andvicine.
 13. The immunochromatography according to claim 3, wherein theneutralization solution contains HCl and at least one selected from thegroup consisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide,and vicine, or contains NaOH and at least one selected from the groupconsisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide, andvicine.
 14. The immunochromatography according to claim 4, wherein theneutralization solution contains HCl and at least one selected from thegroup consisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide,and vicine, or contains NaOH and at least one selected from the groupconsisting of tricine, Tris, HEPES, acetamidoglycine, glycinamide, andvicine.
 15. The immunochromatography according to claim 1, wherein aparticle diameter of the magnetic particle before modification is 0.05μm to 10 μm.
 15. The immunochromatography according to claim 1, whereina particle diameter of the magnetic particle before modification is 0.05μm to 10 μm.
 16. The immunochromatography according to claim 2, whereina particle diameter of the magnetic particle before modification is 0.05μm to 10 μm.
 17. The immunochromatography according to claim 3, whereina particle diameter of the magnetic particle before modification is 0.05μm to 10 μm.
 18. The immunochromatography according to claim
 4. whereina particle diameter of the magnetic particle before modification is 0.05μm to 10 μm